The mucosae of the oral, nasopharyngeal, gastrointestinal, respiratory, and vaginal tracts are the points of entry for most infectious microorganisms. To preclude infection by those agents, which include multiple antibiotic resistant microorganisms (e.g., Staph aureus, Strep. spp., etc.) and pathogens that are newly emerging or reemerging (e.g., Ebola virus, enterovirus, tuberculosis, Clostridium difficile, Cryptococcus gattii, SARS, avian flu, etc.), vaccines that will induce strong and protective mucosal responses are desperately needed. Unfortunately, endogenous regulatory systems that suppress immune responses to foreign antigens make it challenging to elicit strong immune responses to vaccines on the mucosae. To circumvent these suppressive systems, vaccines must be administered in the presence of mucosal adjuvants. To date, the most potent mucosal adjuvants that have been described belong to the superfamily of bacterial heat-labile enterotoxins (HLT). Recently, it was determined that LT, a prototypical HLT of the Type I HLT subfamily, when introduced via the intranasal route, has a propensity to traffic from the nasal cavity to the brain via the olfactory nerve where the HLT induces expression of inflammatory cytokines, an effect that is likely initiated by binding to its specific ganglioside receptor. This undesirable side-effect has hindered the clinical use of HLTs as mucosal adjuvants. To abrogate this undesirable side-effect and to restore the potential use of HLTs as mucosal adjuvants for clinical use, our laboratory has evaluated the immune properties of LT-IIb(T13I), a single point substitution mutant of a non-human Type II HLT that has: (i) altered affinity for its ganglioside receptors, (ii) no detectible toxicity, (iii) strong mucosal adjuvant properties, but (v) DOES NOT induce neuroinflammation. Two additional non-toxic HLTs have been engineered that exhibit similar receptor and immune properties. We HYPOTHESIZE that intranasally administered LT-IIb(T13I), LT-IIb(T14I), and LT- IIb(T13I/T14I), due to their altered receptor-binding properties, do not mobilize into the brain, or alternatively, do not induce expression of neuroinflammatory cytokines or recruit inflammatory cells into the brain. We will test that hypothesis using radioiodinated HLTs, immunohistochemistry, and In Vivo Expression Imaging (IVET). Flow cytometry will be employed to evaluate the cytokine environment and the presence or absence of inflammatory cells in the brains of mice intranasally administered mutant and wt HLTs. While we recognize the current resistance to evaluating HLTs as adjuvants in clinical trials, these pre-clinical experiments will demonstrate the safely and efficacy of this new class o mutant HLT adjuvants. At the successful completion of this proof-of-principle research, we will submit an R01 application to investigate the properties of the mutant HLTs in non-human primates, which will provide the data needed to move these valuable immune agents into clinical trials and, ultimately, into clinical use.